Clostridium difficile is a Gram positive, spore forming anaerobic bacillus, and many strains of this species have acquired resistance to a majority of commonly used antibiotics. The reduction of commensal microflora as an effect of use of antibiotics allows C. difficile to grow and to produce harmful toxins in the intestine, without nutritional competition from normal bacterial flora. Transmitted primarily through contact with contaminated surfaces, C. difficile is a common cause of nosocomial antibiotics-associated diarrhea (CDAD) and pseuodomembranous colitis. (Cloud, J. et al. 2007 Cur Opin Gastroenterol 23:4-9). Infection caused by C. difficile accounts for millions of patient cases and billions of dollars yearly in treatment in hospitals, nursing homes and other care centers. (O'Brien, et al. November 2007 Infection Control and Hospital Epidemiology 28(11): 1219-1227). Highly virulent strains of C. difficile result in increased incidence of illness and more severe effects in patients. (McDonald, et al. 2005 N Engl J Med. 353:2466-2441).
The diagnosis of C. difficile infection remains a challenge (Wilkins T D et al. 2003 J Clin Microbiol 41:531-4). The current diagnostic modalities mainly consist of the detection of the C. difficile organisms and of their toxins in fecal samples. Isolation of C. difficile from stool culture is seldom carried out clinically because it is labor-intensive and time-consuming (Bartlett J G. 2006 Ann Intern Med 145:758-64). One method commonly used is the detection of the enzyme glutamate dehydrogenase (GDH) of C. difficile, but this approach cannot distinguish the toxigenic strains from non-toxigenic ones. Other methods, such as real-time PCR for detecting bacterial genes, are under evaluation for the diagnosis of C. difficile-associated disease (Peterson L R et al. 2007 Clin Infect Dis 45:1152-60), but require sophisticated equipment and training. These assays, which detect the organism, are associated with an inherent problem in that 10% to 30% of hospitalized patients are colonized with toxigenic or non-toxigenic C. difficile without disease (McFarland L V et al. 1989 N Engl J Med 320:204-10). It is therefore more desirable to detect toxins which are thought to be the cause of C. difficile-associated diarrhea (CDAD) (Kelly C P et al. 2008 N Engl J Med 359:1932-40). The widely used enzyme immunoassays (EIAs) are based on monoclonal antibodies (MAbs) that recognize TcdA and/or TcdB. EIAs are rapid and easy, but suffer from low to moderate sensitivity (Planche T et al. 2008 Lancet Infect Dis (12):777-84). The cytotoxin B assay is the “gold standard” for the laboratory diagnosis of C. difficile infection due to its high sensitivity and specificity (Chang T W et al. 1979 J Infect Dis 140:765-70). The assay mainly detects the presence of TcdB, which is far more potent than TcdA in causing cytopathic changes in cultured cells. The drawbacks of cytotoxin B assay are technical complexity, slow turnaround time (24-72 h) and the requirement for a cell culture facility (Chang T W et al. 1979 J Infect Dis 140:765-70). Given the dramatic increase of cases and severity of CDAD in recent years, a rapid and easy to perform assay with high sensitivity and specificity for the diagnosis of C. difficile infection is an urgent need.
C. difficile produces two major protein toxins, toxin A (TcdA) and toxin B (TcdB), which are 308 kD and 269 kD respectively in size. (U.S. Pat. No. 5,098,826, Wilkins et al., issued Mar. 24, 1992). The two toxins belong to the large clostridial cytotoxin (LCT) family and share 49% amino acid identity. (Just, I. et al. 2004 Reviews of physiology, biochemistry and pharmacology 152:23-47). The toxins have similar structures and share putative receptor binding, transmembrane, and enzymatic domains. (Schirmer, J. et al. 2004 Biochimica et Biophysica acta 1673:66-74). After receptor-mediated internalization and intracellular cleavage, the toxins glucosylate members of the Rho-Rac family of small GTPases at a specific threonine residue in host intestinal epithelial cells, leading to alterations in the actin cytoskeleton, massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa. TcdA is an enterotoxin and has minimal cytotoxic activity, whereas TcdB is a potent cytotoxin with limited enterotoxic activity in animals. The extensive damage to the intestinal mucosa was thought to be primarily attributable to the action of TcdA. However, TcdA and TcdB were found to act synergistically in the intestine. (U.S. Pat. No. 6,939,548, Wilkins et al., issued Sep. 6, 2005).
Currently, C. difficile toxins have heretofore been purified mainly from culture supernatants of toxigenic bacteria; the process presents a number of problems including cumbersome purification methods and the presence of contaminants in the resulting proteins. Production of recombinant C. difficile toxins in Escherichia coli have been attempted without success. Assays for detection of C. difficile are currently commercially available, including antigenic detection of a common bacterial enzyme, glutamate dehydrogenase (GDH), a method that does not differentiate between toxigenic and non-toxigenic strains. Enzyme Immunoassays (EIAs) for detecting TcdA and/or TcdB in stool samples are widely used. However, many immunoassay kits have limited sensitivity or are capable of detecting only one of the toxins. (Novak-Weekly, S. M. et al. 2008 Clin Vaccine Immunol; Russman, H. et al. 2007 Eur J Clin Microbiol Infect Dis 26(2): p. 115-9; Staneck, J. L. et al. 1996 J. Clin Microbiol. 34(11): p. 2718-21; Whittier, S. et al. J. Clin Microbiol. 31(11): p. 2861-5; Barbut, F., et al. J Clin Microbiol. 31(4): p. 963-7). Disadvantages of the cytotoxin B assay (CBA) that detects TcdB include technical complexity and slow turnaround time, restrict routine clinical use of the assay for diagnosis. Further, sensitivity of CBA for TcdA is very low.
The growing incidence and severity of C. difficile indicate a need for better understanding of pathogenesis in patients, and development of new assay and treatment tools and methods to obtain relatively pure and biologically active TcdA and TcdB for research. It is desirable to obtain relatively pure and biologically active TcdA and TcdB for studying the pathogenesis of CDAD and host immune response to the infection and for generating immunological tools for research and clinical diagnosis. The native toxins are usually purified from toxigenic C. difficile culture supernatant, which involves multiple steps and the purity is often unsatisfactory (Krivan H C et al. 1987 Infect Immun 55(8):1873-1877; Sullivan N M et al. 1982 Infect Immun 35(3):1032-1040; Keel M K et al. 2007 Veterinary pathology 44(6):814-822). Attempts have been made to clone and express C. difficile toxins in Escherichia coli (Phelps C J et al. 1991 Infection and immunity 59(1):150-153; Tang-Feldman Y J et al. 2002 Molecular and cellular probes 16(3):179-183; Wren B W et al. 1987 FEBS letters 225(1-2):82-86), but it is unclear whether or not purified toxins were obtained from the bacterial lysate in these studies. The Gram-positive Bacillus megaterium expression system may offer an alternative for the expression of C. difficile toxins due to several advantages over the E. coli system, including the lack of alkaline proteases activity and endotoxin liposaccharides (LPS), and the ability to secrete expressed heterologous protein into the medium (Malten M et al. 2006 Applied and environmental microbiology 72(2):1677-1679; Vary P S et al. 2007 Applied microbiology and biotechnology 76(5):957-967). Burger et al. expressed purified recombinant TcdA in B. megaterium however obtained only low levels of expression (Burger S et al. 2003 Biochem Biophys Res Commun 307(3):584-588)). In examples herein full-length proteins of both TcdA and TcdB in B. megaterium were expressed, and an average of 5-10 mg of highly purified recombinant proteins from one liter of total bacterial culture was obtained. Both recombinant TcdA and TcdB were biologically active similar to their native purified toxins.